Abstract
The mechanical microenvironment serves as an important factor influencing stem cell differentiation. Mechanobiological responses depend strongly on actomyosin contractility and integrin-based cell-extracellular matrix (ECM) interactions mediated by adhesive structures such as focal adhesions (FAs). While the roles of FAs in mechanobiology have been intensively studied in many mesenchymal and migratory cell types, recently it has been recognized that certain pluripotent stem cells (PSCs) exhibited significantly attenuated FA-mediated mechanobiological responses. FAs in such PSCs are sparsely distributed and much less prominent in comparison to "classical" FAs of typical adherent cells. Despite these differences, insights into how FAs in PSCs are structurally organized to perform their functions are still elusive. Using mouse embryonic stem cells (mESCs) to study PSC-ECM interactions, here we surveyed the molecular composition and nanostructural organization of FAs. We found that, despite being small in size, mESC FAs appeared to be compositionally mature, containing markers such as vinculin, zyxin, and α-actinin, and dependent on myosin II contractility. Using super-resolution microscopy, we revealed that mESC FAs were organized into a conserved multilayer nanoscale architecture. However, the nanodomain organization was compressed in mESCs, with the force transduction layer spanning ∼10 nm, significantly more compact than in FAs of other cell types. Furthermore, we found that the position and orientation of vinculin, a key mechanotransduction protein, were modulated in an ECM-dependent manner. Our analysis also revealed that while most core FA genes were expressed, the expression of LIM domain proteins was comparatively lower in PSCs. Altogether our results suggest that while core structural and mechanosensitive elements are operational in mESC FAs, their structural organization and regulatory aspects may diverge significantly from "classical" FAs, which may account for the attenuated mechanobiological responses of these cell types.